1. Field of the Invention
This invention relates to a suppression of carcinoma using a high purity conjugated fatty acid provided by a novel synthesis.
2. Background
Conjugated linoleic acid (CLA) is a general term used to name positional and geometric isomers of linoleic acid.
Conjugated eicosadienoic acid (CEA) is a general term used to name positional and geometric isomers of the C-20 fatty acid of 11-cis, 13-trans eicosadienoic acid, also known as 11(Z), 13(E)-eicosadienoic acid.
Linoleic acid and eicosadienoic acid are straight chain carboxylic acids having double bonds between the ninth and tenth, twelfth and thirteenth carbons and eleventh and twelfth, fourteenth and fifteenth carbons, respectively. Linoleic acid is 9-5 cis, 12-cis octadecadienoic acid [9(Z),12(Z)-octadecadienoic acid]. The numbers are counted from the carboxylic acid moiety. See Formula (1) for 9-cis, 12-cis octadecadienoic acid [9(Z),12(Z)-octadecadienoic acid]. See Formula (2) for 11-cis, 13-trans eicosadienoic acid, [11(Z),13(E)-eicosadienoic acid]. 
Conjugated linoleic acid (CLA) has two conjugated double bonds between the ninth and the twelfth carbons or between the tenth and thirteenth carbons, with possible cis and trans combinations. Conjugated eicosadienoic acid (CEA) has two conjugated double bonds between the eleventh and fourteenth or between the twelfth and fifteenth carbons, with possible cis and trans combinations. Conjugated double bonds means two or more double bonds which alternate in an unsaturated compound as in 1,3 butadiene. The hydrogen atoms are on the same side of the molecule in the case of cis. The hydrogen atoms are on the opposite side of the molecule in the case of trans. See Formula (3) for conjugated linoleic acid (CLA). See Formula (4) for conjugated eicosadienoic acid (CEA). 
The free, naturally occurring conjugated linoleic acids (CLA) have been previously isolated from fried meats and described as anticarcinogens by Y. L Ha, N K. Grimm and M. W. Pariza, in Carcinogenesis, Vol. 8, No. 12, pp. 1881-1887 (1987). Since then, they have been found in some processed cheese products (Y. L. Ha, N. K. Grimm and M. W. Pariza, in J. Agric. Food Chem., Vol. 37, No. 1, pp. 75-81 (1987)).
The free, naturally occurring conjugated eicosadienoic acids (CLA) are not known to exist.
Cook et al. in U.S. Pat. No. 5,554,646 disclose animal feeds containing CLA, or its non-toxic derivatives, e.g., such as the sodium and potassium salts of CLA, as an additive in combination with conventional animal feeds or human foods. CLA makes for leaner animal mass.
Conjugated linoleic acid (CLA) has a significant potency relative to other fatty acids in respect to an ability to modulate tumorigenisis. Conjugated linoleic acid (CLA) is closely related to linoleic acid but differs from linoleic acid in the position and configuration of the double bonds. Linoleic acid has a stimulatory effect on Carcinogenesis, as contrasted with the ability of conjugated linoleic acid (CLA) to inhibit tumor development. In this way, conjugated linoleic acid (CLA) has an opposite effect as contrasted with linoleic acid in treating carcinoma.
Further comparing the ability of conjugated linoleic acid (CLA) relative to other fatty acids in respect to an ability to modulate tumorigenisis, eicosapentaenoic acid and docosahexaenoic are the major components in fish oil which are responsible for tumor suppression. However, the amount of fish oil needed to suppress tumors will exceed 10% in the diet.
Adding conjugated linoleic acid (CLA) in respect to an ability to modulate tumorigenisis is contrasted with a method of reducing fat intake, nonspecific fat intake, to inhibit tumors.
Accordingly, conjugated linoleic acid (CLA) has a significant potency relative to other fatty acids in respect to an ability to modulate tumorigenisis.
The free acid forms of the CLA may be prepared by isomerizing linoleic acid. The terms xe2x80x9cconjugated linoleic acidsxe2x80x9d and xe2x80x9cCLAxe2x80x9d as used herein are intended to include 9,11-octadecadienoic acid, 10,12-octadecadienoic acid, mixtures thereof, and the non-toxic salts of the acids. The non-toxic salts of the free acids may be made by reacting the free acids with a non-toxic base.
Historically, CLA was made by heating linoleic acid in the presence of a base. The term CLA (conjugated linoleic acid) refers to the prior art preparation involving alkali cooking of linoleic acid.
A conventional method of synthesizing CLA is described in Example I. However, CLA may also be prepared from linoleic acid by the action of a linoleic acid isomerase from a harmless microorganism, such as the Rumen bacterium Butyrivibrio fibrisolvens. Harmless microorganisms in the intestinal tracts of rats and other monogastric animals may also convert linoleic acid to CLA (S. F. Chin, J. M. Storkson, W. Liu, K. Albright and M. W. Pariza 1994, J. Nutr., 124; 694-701).
The prior art method of producing conjugated linoleic acids (CLA) can be seen in the following Example I using starting materials of linoleic acid or safflower oil.
Ethylene glycol (1000 g) and 500 g potassium hydroxide (KOH) are put into a 4-neck round bottom flask (5000 ml). The flask is equipped with a mechanical stirrer, a thermometer, a reflux condenser, and a nitrogen inlet. The nitrogen to be introduced is first run through two oxygen traps.
Nitrogen is bubbled into the ethylene glycol and KOH mixture for 20 minutes, and the temperature is then raised to 180xc2x0 C.
1000 g of linoleic acid, corn oil, or safflower oil is then introduced into the flask. The mixture is heated at 180xc2x0 C. under an inert atmosphere for 2.5 hours.
The reaction mixture is cooled to ambient conditions, and 600 ml HCL are added to the mixture which is stirred for 15 minutes. The pH of the mixture is adjusted to pH 3. Next, 200 ml of water is added into the mixture and stirred for 5 minutes. The mixture is transferred into a 4 L separatory funnel and extracted three times with 500 ml portions of hexane.
The aqueous layer is drained, and the combined hexane solution is extracted with four 250-ml portions of 5% NaCl solution.
The hexane is washed 3 times with water. The hexane is transferred to a flask, and the moisture in the hexane is removed with anhydrous sodium sulfate (Na2SO4). The hexane is filtered through Whatman paper into a clean 1000 ml round bottom flask, and the hexane is removed under vacuum with a rotoevaporator to obtain the CLA. The CLA is stored in a dark bottle under argon at xe2x88x9280xc2x0 C. until time of use.
The CLA obtained by the practice of the described prior art methods of preparation typically contains two or more of the 9,11-octadecadienoic acids and/or 10-12-octadecadienoic acids and active isomers thereof. After alkali treatment, the compound may be in the free acid or salt form. The CLA is heat stable and can be used as is, or it may be dried in a solvent. The CLA is readily converted into a non-toxic salt, such as the sodium or potassium salt, by reacting chemically equivalent amounts of the free acid with an alkali hydroxide at a pH of about 8 to 9.
Theoretically, eight (8) possible geometric isomers of 9,11 and 10,12-octadecadienoic acid (c9,c11; c9,t11; t9,c11; t9,t11; c10, c12; c10, t12; t10, c12; and t10, t12) would form from the isomerization of c9, c12 octadecadienoic acid. As a result of the isomerization, only four isomers (c9, c11; c9, t11; t10, c12; and c10, c12) would be expected. However, of the four isomers, c9, t11- and t10, c12-isomers are predominantly produced during the autoxidation or alkali isomerization of c9, c12-linoleic acid because of the co-planar characteristics of 5 carbon atoms around a conjugated double bond and spatial conflict of the resonance radical. The remaining two c,c-isomers are minor contributors as are the other isomers. Because of double bond shifts, more isomers are produced. Positional isomers have been found for a total of twelve isomers identified so far.
The relatively higher distribution of the t,t-isomers of 9,11- or 10,12-octadecadienoic acid apparently results from the further stabilization of c9, t11- or t10, c12-geometric isomers, which is thermodynamically preferred, during an extended processing time or long aging period. Additionally, the t,t-isomer of 9,11- or 10,12-octadecadienoic acid predominantly formed during the isomerization of linoleic acid geometrical isomers (t9,t12-, c9,t12-, and t9,c12-octadecadienoic acid) may influence the final ratio of the isomers or the final CLA content in the samples.
Linoleic acid geometrical isomers also influence the distribution of minor contributors (c,c-isomers of 9,11- and 10,12-, t9,c11- and c11,t12-octadecadienoic acids). The 11,13-isomer might be produced as a minor product from c9,c12-octadecadienoic acid or from its isomeric forms during processing.
Conjugated linoleic acid (CLA) has long been of interest to biochemists and nutritionists. A recent article in INFORM, Vol. 7, No. 2, Feb. 1996, published by the American Oil Chemists"" Society summarizes some of the data developed so far. The article stresses the feed use for which the product is currently being developed, resulting in less fat and more lean meat in animals. A number of other recent articles stress effects in fighting cancer. In many cases, one isomer, 9(Z),11(E)-CLA, has been named as the active isomer, mainly because it alone is incorporated into the phospholipids of the organism being fed CLA.
CLA has been shown to have preventive effects on breast cancer in mice. CLA is not used for humans today, mostly because it is not available except in impure forms. CLA is not approved by the FDA, and impurities can have a detrimental influence on toxicity tests to obtain FDA approval.
The problem with CLA, as it is available today, has been the fact that only a diverse mixture of isomers can be made. Conventional synthesis methods involve the isomerization of linoleic acid by potassium hydroxide at about 200xc2x0 C. This procedure yields about equal amounts of the 9,11- and 10,12-isomers which are almost impossible to separate. The content of the preferred isomer of 9(Z),11(E)-CLA in the mix is about 20-30%. All of the isomers presumed to be in the mix have been synthesized but only by very laborious methods that are quite unsuitable for large scale manufacture.
Heating the linoleic acid in the presence of a base such as alkali, makes the double bond move over, and it does so in a haphazard way. The geometry changes, and the resultant product is the 9-cis, 11-trans isomer in a yield of only 23-40%.
It is an object of the present invention to provide a method for treatment of carcinoma in a human using a purified conjugated fatty acid produced by a novel synthesis to provide over 50 percent of a fatty acid having somewhere in its carbon chain a chain of four carbons with two conjugated double bonds.
It is an object of the present invention to provide a method for treatment of carcinoma in a human using a purified conjugated fatty acid produced by a novel synthesis to provide over 70 percent of a fatty acid having somewhere in its carbon chain a chain of four carbons with two conjugated double bonds.
It is an object of the present invention to provide a method for treatment of carcinoma in a human using a higher purity conjugated fatty acid than is available from conventional sources containing impurities which may have a detrimental influence on toxicity tests to obtain FDA approval.
It is an object of the present invention to provide a method for treatment of carcinoma in a human using a higher purity conjugated fatty acid than is available from conventional sources containing diverse mixtures of isomers which may have a detrimental influence on toxicity tests to obtain FDA approval.
These and other objects of the present invention will be described in the detailed description of the invention which follows. These and other objects of the present invention will become apparent to those skilled in the art from a careful review of the detailed description and from reference to the figures of the drawings.
The present invention provides a method for the treatment of carcinoma in a human, including administering to a human a therapeutically effective amount of a conjugated fatty acid.
The present invention provides a synthesis for producing conjugated fatty acid. A fatty acid ester is selected having somewhere in its carbon chain a chain of four carbon atoms such that carbon one bears one hydrogen and one hydroxyl group, carbon two bears two hydrogen atoms, and a double bond is positioned between carbon three and carbon four. The fatty acid ester is reacted with a chloride including toluenesulfonyl chloride, methanesulfonyl chloride, benzenesulfonyl chloride, alkylsulfonyl chloride, or arylsulfonyl chloride at a temperature in the range of 10-100xc2x0 C. to form an alkyl- or aryl-sulfonyl ester. The fatty acid bearing the alkyl- or aryl-sulfonyl ester is reacted with diazabicyclo-undecene to form a conjugated fatty acid ester having a purity greater than 40%. The conjugated fatty acid is formed in high yield. In one aspect, the sulfonyl ester of a fatty acid ester is formed in a pyridine solvent. In one aspect, the sulfonyl ester of a fatty acid ester is reacted with diazabicyclo-undecene in a polar, non-hydroxylic solvent acetonitrile.
The present invention provides a method for the treatment of carcinoma in a human, including administering to human a purified conjugated fatty acid produced by the novel room temperature synthesis process of the present invention.